A hot start is beneficial or necessary for efficiency and reliability of a large portion of PCR reactions. The most demanding PCR reactions are especially dependent on a hot start. A heat switch can be added to PCR protocols by any of several manual or built-in methods, all of which have drawbacks, such as narrow conditions, extra expense, extra steps, prone to error, prone to cross-contamination, and/or lack of applicability to long PCR. We proposed to develop a Taq DNA polymerase mutant that is heat-switchable, which is much less active than the wild-type at room (reaction setup) temperature, yet normally active at 65-70 degrees, and still resistant to 95 degrees. In Phase I we were able to isolate and initially characterize several mutants of Klentaq DNA polymerase that outperformed the parent enzyme in hot start PCR. We will test single amino acid changes found in these mutants to identify the ones that confer cold-sensitivity. We also plan to optimize the amino acid substitutions for each functionally significant mutation site. The best mutants will also be used to engineer cold-sensitive Taq DNA polymerase. Finally, we will purify mutant and combined-mutant enzymes to test their advantages in demanding PCR (short and long) that requires a hot start under a broad range of conditions. PROPOSED COMMERCIAL APPLICATIONS: Hot-start methods for PCR more and more important to efficiency and reproducibility. An estimated 10-15% of the assays performed employ one of the current methods, with cost being a significant factor. Benefitting the most from hot start are forensic and diagnostic applications of PCR. Other important benefits are food and blood contamination PCR assays. All of these assays are rapidly growing areas and could benefically employ the proposed enzyme.